|Cell plating step
- Use the volume recommended by the manufacturer to plate transformed cells.
- Adjust cell dilutions as necessary to obtain the desired number of colonies.
- Recover the cells in enriched medium (e.g., S.O.C medium) after heat shock and allow sufficient time for growth (e.g., 1 hour) before plating.
- Make sure that the antibiotic used in the media is the same as the vector’s resistance marker.
|Antibiotic concentration is too high
|Bacterial transformation step
|Poor transformation efficiency
- Check the competency of your cells using 0.1 ng of an intact, supercoiled vector DNA (e.g., pUC19) in a transformation reaction. The competent cells should yield at least 1 x 106 transformants per µg of supercoiled DNA. This corresponds to approximately 100 colonies when using 0.1 ng of uncut plasmid for a transformation.
- For longer inserts (e.g., >5 kb), consider electroporation instead of chemical transformation to increase transformation efficiency.
- For chemical transformation, choose competent cells with higher transformation efficiencies (>1 x 109 CFU/μg) or those specifically tested to handle large plasmids.
- See also transformation troubleshooting guide.
- Some cloned sequences may not be tolerated by the host E. coli strain. Check the target sequence for strong E. coli promoters and for inverted repeats. If the protein expressed by the cloned sequence is toxic to E. coli, try using promoters that have low level expression or that are inducible with tight regulation. Also consider using a low copy number plasmid as a cloning vehicle.
- Try a different strain that tolerates repeated sequences (e.g., Stbl2 E. coli).
- Try growing the cells at a lower temperature (30°C or room temperature). Colonies will take longer to appear.
|Excess ligase in transformation mixture
- The presence of T4 DNA ligase can reduce transformation efficiencies. Do not use more than 5 µL of ligation mixture for 50 µL of competent cells or 1 µL for electrocompetent cells.
|Poor ligation efficiency
- Perform a control reaction to check that the ligase remains active. Optimize ligation parameters, such as insert:vector ratios, reaction time, and reaction temperature, to maximize insertion of the desired fragment.
|Poor DNA quality
- Ensure the insert and vector DNA fragments are free of remnants of the digestion reaction (e.g., active restriction enzymes) or contaminants (e.g., excess salts, EDTA, phenol, etc.) that may inhibit the ligation reaction. Purify the vector and insert by gel electrophoresis, column purification, or phenol:chloroform:iso-amyl solvent extraction prior to ligation.
|Ligase carried over to transformation mixtures
- The presence of T4 DNA ligase can reduce transformation efficiencies. Do not use more ligase than recommended in the ligation reaction. If necessary, inactivate T4 DNA ligase by chloroform extraction, heat inactivation, or spin-column purification before transformation.
|Incompatible ends in ligation
- Review overhangs generated by the chosen restriction enzymes for compatibility.
|Fragment preparation step
|Lack of 5′ phosphate
- In blunt-end cloning, the overhangs generated by the restriction enzymes may have been inefficiently filled-in and/or trimmed. Use optimal blunt-end methods for the overhangs.
- DNA may have been damaged by UV light during excision from agarose gel. To minimize UV-induced damage, use a long wavelength UV (360 nm) light box when excising DNA from gel and limit exposure time as much as possible.
- If using a short wavelength (254–312 nm) light box, limit exposure of the DNA to a few seconds and keep the gel on a glass or plastic plate during illumination.
- Alternatively, use dyes with long excitation (less damaging) wavelengths to visualize the DNA.
- Another option is to load the DNA sample in two or more lanes. After electrophoresis, cut away and stain only one lane with ethidium bromide. Use this stained lane as a reference for excising the DNA from the unstained lanes.
|Restriction digestion step
|Unexpected or incomplete cleavage
- Ensure restriction enzymes are free of contaminating endonucleases, exonucleases, or phosphatases that may damage the DNA ends. Use enzymes that are produced under highest quality standards for cloning.
- Perform restriction digestion as recommended by the enzyme supplier. Check for reaction conditions, buffer compatibility, and required cofactors for optimal digestion.
- Check DNA fragments by gel electrophoresis after restriction digestion to assess the correct band sizes, as well as any unexpected cleavage such as smears and extra bands.
- Sequence the digested fragments to check the cut sites, if necessary.